CS1-specific chimeric antigen receptor engineered immune effector cells

ABSTRACT

Disclosed herein are chimeric antigen receptors (CAR) that can specifically recognize tumor-associated antigens (TAA) on multiple myeloma (MM) cells. Also disclosed are immune effector cells, such as T cells or Natural Killer (NK) cells, that are engineered to express these CARs. Therefore, also disclosed are methods of providing an anti-tumor immunity in a subject with MM that involves adoptive transfer of the disclosed immune effector cells engineered to express the disclosed CARs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/888,877 filed Nov. 3, 2015, which is a National Phase Applicationof PCT/US2014/036684 filed May 2, 2014, which claims benefit of U.S.Provisional Application No. 61/819,141, filed May 3, 2013, and U.S.Provisional Application No. 61/876,492, filed Sep. 11, 2013, which arehereby incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted May 17, 2017, as a text file named“10336-088US2 2017_05_17 Sequence_Listing.txt,” created on May 17, 2017,and having a size of 25,185 bytes is hereby incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

BACKGROUND

Multiple myeloma (MM) is a B-cell malignancy characterized by theaberrant clonal expansion of plasma cells (PCs) within the bone marrow,with an estimated 21,700 new cases and 10,710 deaths from MM identifiedin the United States in 2012 (Siegel R, et al. Cancer J Clin 201262:10-29). In 2013, it has been estimated that 22,350 individuals willbe newly diagnosed with MM in the United States and 10,710 people willdie from it, accounting for 20% of the deaths from all hematologicmalignancies. Despite the use of proteasome inhibitors andimmune-modulating drugs, which have improved overall survival (PalumboA, et al. Leukemia 2009 23:449-456), MM remains an incurable malignancy(Podar K, et al. Leukemia 2009 23:10-24) for which novel therapeuticapproaches are urgently needed.

SUMMARY

Disclosed herein are chimeric antigen receptor (CAR) polypeptides thatcan be used with adoptive cell transfer to target and kill multiplemyeloma (MM) cells. The cell surface glycoprotein CS1 is highly andubiquitously expressed on the surface of myeloma cells while beingexpressed at very low levels in the majority of immune effector cells.Therefore, the disclosed CAR polypeptides contain in an ectodomain ananti-CS1 binding agent that can bind CS1-expressing MM cells. As withother CARs, the disclosed polypeptides can also contain a transmembranedomain and an endodomain capable of activating an immune effector cell.For example, the endodomain can contain an intracellular signalingdomain and optionally a co-stimulatory signaling region.

The anti-CS1 binding agent is in some embodiments an antibody fragmentor an antigen-binding fragment that specifically binds CS1. For example,the antigen binding domain can be a Fab or a single-chain variablefragment (scFv) of an antibody that specifically binds CS1. The anti-CS1binding agent is in some embodiments an aptamer that specifically bindsCS1. For example, the anti-CS1 binding agent can be a peptide aptamerselected from a random sequence pool based on its ability to bind CS1.The anti-CS1 binding agent can also be a natural ligand of CS1, or avariant and/or fragment thereof capable of binding CS1.

In some embodiments, the intracellular signaling domain is a CD3 zeta(CD3ζ) signaling domain, and the costimulatory signaling regioncomprises the cytoplasmic domain of CD28, 4-1BB, or a combinationthereof. In some cases, the costimulatory signaling region contains 1,2, 3, or 4 cytoplasmic domains of one or more intracellular signalingand/or costimulatory molecules.

Also disclosed are isolated nucleic acid sequences encoding thedisclosed CAR polypeptides, vectors comprising these isolated nucleicacids, and cells containing these vectors. For example, the cell can bean immune effector cell selected from the group consisting of a T cell,a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), and aregulatory T cell. In some embodiments, the cell exhibits an anti-tumorimmunity when the antigen binding domain of the CAR binds to CS1.

Also disclosed is a method of providing an anti-tumor immunity in asubject with multiple myeloma (MM) that involves administering to thesubject an effective amount of an immune effector cell geneticallymodified with a disclosed CS1-specific CAR.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A to 1C show the generation of a CS1-specific CAR and itsexpression in CAR-transduced T cells. FIG. 1A is a schematic diagram ofa Pinco-CS1-CAR retroviral construct containing a scFv against CS1linked to CD28 and CD3ζ endodomains. LTR, long terminal repeat; SP,signal peptide; VH, variable H chain; L, linker; VL, variable L chain.In FIG. 1B, PBMC (peripheral blood mononuclear cells) were activatedwith CD3 and CD28 beads and transduced with the Pinco-CS1-CAR or Pincoconstruct. GFP-positive cells were sorted, and cell lysates weresubjected to immunoblot analysis under reducing conditions withanti-human CD3ζ primary antibody. In FIG. 1C, mock1- orCS1-CAR1-transduced T cells from healthy donors were stained withbiotin-labeled goat anti-mouse Fab-specific or isotype-matched controlantibody, followed by streptavidin and CD3 antibody staining.

FIGS. 2A to 2C show CS1-redirected T cells secrete more IFN-γ and IL-2than mock T cells in response to CS1-expressing myeloma cell lines. FIG.2A shows flow cytometric analysis of CS1 expression on the surface ofmyeloma cell lines. The four myeloma cell lines indicated were stainedwith PE-conjugated anti-CS1 mAb antibody (solid line) or isotype-matchedcontrol antibody (dotted line). FIGS. 2B and 2C are bar graphs showingIFN-γ (FIG. 2B, ng/ml) and IL-2 (FIG. 2C, pg/ml) secretion in mock- orCS1-CAR-transduced healthy donor T cells (2×10⁵) that were culturedalone (no target) or stimulated with an equal number of myeloma cellsexpressing different levels of CS1 for 24 hours.

FIGS. 3A to 3D show CS1-redirected T cells preferentially eradicatemyeloma cells obviously expressing CS1 protein. In FIG. 3A, ⁵¹Cr-labeledNCI-H929, IM9, MM.1S, and RPMI-8226 myeloma cells (5×10³) werecocultured with mock- or CS1-CAR-transduced T cells at the indicated E/Tratios for 4 hours, and target lysis (⁵¹Cr release) was measured. InFIG. 3B, expression of the degranulation marker CD107a and the T-cellactivation marker CD69 on mock- or CS1-CAR transduced T cells wereevaluated by flow cytometry following 4 hours co-culture with NCI-H929cells. Compared with mock-transduced T cells, CS1-CAR-transduced T cellsdisplayed superior degranulation and higher T-cell activation inresponse to CS1-expressing NCI-H929 cells. In FIG. 3C, Mock- andCS1-CAR-transduced T cells were permeabilized for intracellular stainingwith mAb specific for granzyme B and perforin, and analyzed by flowcytometry.

FIGS. 4A to 4D show ectopic overexpression of CS1 in MM cells triggersenhanced cytotoxicity and cytokine secretion after recognition byCS1-CAR T cells. FIG. 4A shows flow cytometric staining for CS1 proteinor IgG isotype control (dotted line) on the surface of RPMI-8226 cellsoverexpressing CS1 (RPMI-8226-CS1, solid heavy line) or an empty vectorcontrol (RPMI-8226-PCDH, solid light line). FIG. 4B is a graph showingcytotoxicity of mock- or CS1-CAR-transduced T cells againstRPMI-8226-CS1 and RPMI-8226-PCDH cells. RPMI-8226-CS1 and RPMI-8226-PCDHcells were incubated with mock- or CS1-CAR-transduced T cells atindicated E/T ratios for 4 hours, and specific lysis was determinedusing a standard ⁵¹Cr release assay. FIGS. 4C and 4D are bar graphsshowing IFN-γ (FIG. 4C, pg/ml) and IL-2 (FIG. 4D, pg/ml) secretion inmock- or CS1-CAR-transduced T cells (1×10⁵) cultured alone or stimulatedwith an equal number of either RPMI-8226-CS1 or RPMI-8226-PCDH cells.

FIGS. 5A to 5D show CS1-CAR T cells specifically recognize and eliminateCS1-expressing human primary myeloma cells ex vivo. FIG. 5A shows flowcytometric results of PBMCs from patients with MM that were activatedwith anti-CD3 and anti-CD28 beads, transduced with the Pinco-CS1-CAR orPinco construct (mock), and stained with anti-mouse Fab andanti-humanCD3 antibodies. Results from 1 of 4 patients with similar dataare shown. FIG. 5B shows flow cytometric staining for CS1 protein inCD138⁺ myeloma cells freshly isolated from patients with MM. Resultsfrom 3 of 10 patients with similar data are shown. FIG. 5C is a seriesof graphs showing specific lysis (⁵¹Cr release assay) of the CD138⁺myeloma cells in (B) co-cultured with the autologous mock- orCS1-CAR-transduced T cells in (A) at indicated E/T ratios for 4 hours.FIG. 5D is a bar graph showing IFN-γ secretion (pg/ml) by the cellstreated as in (C) except that the E/T ratio was 1:1 and the incubationtime was extended to 24 hours.

FIGS. 6A and 6B show CS1-redirected T cells inhibit tumor growth andprolong mouse survival in an orthotopic MM.1S xenograft mouse model.FIG. 6A is a series of dorsal and ventral bioluminescence images of fiverepresentative mice bearing MM.1S tumors from each indicated group. NSGmice were intravenously inoculated with 8×10⁶ MM.1S cells expressingluciferase (day 0). On days 7 and 14 after inoculation, each mousereceived PBS (placebo control group), 10×10⁶ mock T cells (mock controlgroup) or CS1-CAR T cells (CAR treatment group). FIG. 6B showsKaplan-Meier survival curves of MM.1S bearing mice treated with PBS,mock T cells, or CS1-CAR T cells.

FIGS. 7A to 7E show 293T transformed cells expressing CS1 weresusceptible to recognition and lysis by CS1-CAR T cells. FIG. 7A showsthat 293T parental cells were negative for CS1 expression. 293T cellswere stained with PE-conjugated anti-CS1 mAb antibody (solid line) orisotype-matched control Ab (dotted line) and analyzed by flow cytometry.FIG. 7B shows flow cytometric staining for CS1 protein on the surface of293T cells overexpressing CS1 (293T-CS1, dark solid heavy line) or anempty vector (293T-PCDH, gray solid heavy line). 293 T cells expressingCS1 stained with IgG isotype (dotted line) served as non-specificbinding control. FIG. 7C shows cytotoxicity of mock- orCS1-CAR-transduced T cells against 293T-CS1 and 293T-PCDH cells.293T-CS1 and 293T-PCDH cells were incubated with mock- orCS1-CAR-transduced T cells at indicated E/T ratios for 4 h, and specificlysis was determined using a standard ⁵¹Cr release assay. FIGS. 7D and7E are a bar graph showing IFN-γ secretion (FIG. 7D, pg/ml) or IL-2secretion (FIG. 7E, pg/ml) by Mock- or CS1-CAR-transduced T cellscultured alone or stimulated with either 293T-CS1 or 293T-PCDH cells.

FIGS. 8A and 8B show that both CD4⁺ and CD⁸⁺ CS1-CAR T cells wereactivated in response to myeloma cells. In FIG. 8A, Mock- orCS1-CAR-transduced T cells were cultured alone or stimulated withNCI-H929 and MM.1S cells for 12 h, then surface expression of CD3 andCD8, as well as intracellular IFN-γ, were evaluated by flow cytometry.The plots were gated on CD3+ lymphocytes. One representative experimentout of three with similar results is shown. In FIG. 8B, Mock- orCS1-CAR-transduced T cells were cultured alone or stimulated withNCI-H929 and MM.1S cells for 4 h, and expression of CD3, CD8 and thedegranulation marker CD107a were evaluated by flow cytometry. The plotswere gated on live CD3⁺ lymphocytes. One representative experiment outof three with similar results is shown.

FIGS. 9A and 9B show that CS1-redirected T cells inhibit tumor growthand prolong mouse survival in an orthotopic IM-9 xenograft mouse model.FIG. 9A show dorsal and ventral bioluminescence images of fiverepresentative mice bearing IM9 tumors from each indicated group. NSGmice were i.v. inoculated with 5×10⁵ IM9 cells expressing luciferase(day 0). On day 7 and day 21 after inoculation, each mouse received PBS(placebo control group), 10×10⁶ mock T cells (mock control group) orCS1-CAR T cells (CAR treatment group). The white crosses “+” representmice that died of MM disease in the PBS-treated group at the time ofimaging. FIG. 9B are Kaplan-Meier survival curves of IM9-bearing micetreated with PBS, mock T cells or CS1-CAR T cells.

FIGS. 10A to 10C show that CS1-CAR T cells persisted and proliferated inthe bone marrow (BM) of MM.1S cell-grafted NSG mice. NSG mice wereinoculated with 8×10⁶ MM.1S cells on day 0, and on day 7 mice weretreated with 10×10⁶ CS1-CAR T cells. On day 20, mice were i.p. injectedwith 1.5 mg Brdu in DPBS solution. Mice were sacrificed on the followingday, and the BM cells were isolated for surface staining with CD45 andCD3 human-specific antibodies and/or anti-Brdu antibodies (BDBiosciences) following the manufacture's protocol. FIG. 10A shows thepercentage of human T cells (CD45⁺/CD3⁺) in the BM of representativemice. In FIG. 10B, the gated human T cells (CD45⁺/CD3⁺) were stainedwith IgG (left panel) or anti-Fab Ab (right panel) to verify CARexpression. In FIG. 10C, the gated human T cells (CD45⁺/CD3⁺) werestained with IgG (left panel) or anti-Brdu Ab, and the percentage ofBrdu-incorporated T cells are displayed.

FIGS. 11A to 11C shows CS1-CAR T cells displayed low levels ofreactivity against primary NK and T cells. ⁵¹Cr-labeled human primary NKand T cells (5×10³) were co-cultured with mock- or CS1-CAR-transduced Tcells at the indicated Effector/Target (E/T) ratios for 4 h, and targetlysis (⁵¹Cr release) of NK cells (FIG. 11A) and T cells (FIG. 11B) wasmeasured. FIG. 11C is a bar graph showing IFN-γ secretion by Mock- orCS1-CAR-transduced T cells cultured alone or stimulated with eitherprimary NK cells, T cells or myeloma cells for 24 h.

FIG. 12 shows primary NK cells or T cells did not trigger apparentactivation induced cell death (AICD) in CS1-CAR T cells. Primary NKcells and T cells were incubated with an equal number of ⁵¹Cr-labelledmock- or CS1-CAR-transduced T cells for 12 h. Specific lysis was thendetermined using a ⁵¹Cr release assay.

FIGS. 13A to 13C show the generation of a CS1-specific CAR and itsexpression in CAR-transduced NK cells. FIG. 13A is a schematicrepresentation of the CS1-CAR lentiviral construct. FIG. 13B showsWestern blotting analysis of CS1-CAR expression using a CD3ζ-specificAb. Data shown are representative of three experiments with similarresults. FIG. 13C shows expression of chimeric CS1 scFv on the surfaceof FACS-sorted NK-92 and NKL cells transduced with the CS1-CAR constructor empty vector (EV) analyzed by flow cytometry after cells were stainedwith an anti-myc antibody or an IgG1 isotype control. Data shown arerepresentative of three experiments with similar results.

FIGS. 14A to 14D show CS1-CAR NK cells eradicate CS1⁺ but not CS1⁻ MMcells. FIG. 14A shows determination of CS1 expression on the surface ofL363, IM9 and U266 MM cell lines by flow cytometry after cells werestained with anti-CS1 mAb or isotype-matched control antibody. FIGS. 14Bto 14D show cytotoxic activity of mock-transduced or CS1-CAR-transducedNK-92 or NKL cells against IM9 (FIG. 14B), L363 (FIG. 14C) and U266(FIG. 14D) cells using a standard ⁵¹Cr release assay. NK-92-EV andNKL-EV indicate empty vector (EV) control-transduced NK-92 and NKLcells, respectively. NK-92-CS1-CAR and NKL-CS1-CAR indicate transductionof NK-92 and NKL cells, respectively, with a CS1-CAR construct. * and **indicate P<0.05 and P<0.01, respectively.

FIGS. 15A to 15C show recognition of CS1⁺ MM cells induces a strongerresponse from CS1-CAR NK cells than from control NK cells. FIGS. 15A to15C are bar graphs showing IFN-γ secretion by Mock-transduced or CS1-CARtransduced NK-92 or NKL effector cells co-cultured with an equal numberof IM9 (FIG. 15A), L363 (FIG. 15B) or U266 (FIG. 15C) myeloma cells for24 h. NK-92-EV and NKL-EV indicate empty vector (EV) control-transducedNK-92 and NKL cells, respectively. NK-92-CS1-CAR and NKL-CS1-CARindicate transduction of NK-92 and NKL cells, respectively, with aCS1-CAR construct.

FIGS. 16A to 16C show enhanced target recognition of NK-92-CS1-CAR cellsdepends on expression of CS1 on MM cells. FIG. 16A shows flow cytometricstaining for a CS1 protein or an IgG control (dotted line) on thesurface of U266 cells overexpressing CS1 (U266-CS1, solid heavy line) oran empty vector control (U266-Vector, solid light line). FIG. 16B showscytotoxicity of mock- or CS1-CAR-transduced NK-92 cells (NK-92-EV andNK-92-CS1-CAR, respectively) against U266-Vector and U266-CS1 cells.U266-Vector or U266-CS1 cells were incubated with NK-92-CS1-CAR orNK-92-EV cells at different Effector/Target (E/T) ratios for 4 h.Specific lysis was determined using a standard ⁵¹Cr release assay. *indicates P<0.05. (c) NK-92-CS1-CAR or NK-92-EV cells were co-culturedwith an equal number of U266-Vector or U266-CS1 myeloma cells for 24 h.Supernatants were then harvested for measurement of IFN-γ secretionusing ELISA.

FIGS. 17A to 17C show phenotypic characterization of CS1-CAR modified NKcells. In FIG. 17A, Mock- or CS1-CAR-transduced NK-92 cells (NK-92-EVand NK-92-CS1-CAR, respectively) were either cultured alone, or culturedwith IM9 MM cells for 4 h. Surface expression of NKp30, NKp46, NKG2C,NKG2D, CD69 and HLA-DR was assessed by flow cytometry following stainingwith the corresponding mAbs, and the mean fluorescence intensity (MFI)was recorded. * indicates P<0.05. In FIG. 17B, NK-92-EV andNK-92-CS1-CAR cells were permeabilized for intracellular staining withmAb specific for perforin or granzyme B, and analyzed by flow cytometry.The dashed line represents staining the NK-92-EV control cells withcontrol IgG antibody, solid heavy line denotes staining NK-92-CS1-CARcells with either Perforin or Granzyme B antibody, and the solid lightline indicates staining the NK-92-EV control cells with either Perforinor Granzyme B antibody. FIG. 17C shows MFI for histograms shown in FIG.17B. * indicates P<0.05.

FIGS. 18A to 18C show CS1-CAR-transduced NK-92 cells enhance killing ofprimary human myeloma cells. FIG. 18A shows flow cytometric staining forCS1 protein or IgG isotype control, demonstrating that CD138⁺ primarymyeloma cells highly express CS1. The open and filled histogramsrepresent staining with isotype-matched control antibodies and anti-CS1antibodies, respectively. Data shown are representative of two out ofsix patient samples with similar results. FIG. 18B shows cytotoxicactivity of mock- or CS1-CAR-transduced NK-92 cells (NK-92-EV andNK-92-CS1-CAR, respectively) against CD138⁺ primary myeloma cells fromthree of six patients with similar results using a standard ⁵¹Cr releaseassay. E/T indicates the effector cell/target cell ratio. * indicatesP<0.05. FIG. 18C shows IFN-γ secretion by CD138⁺ primary myeloma cellsco-cultured with NK-92-EV or NK-92-CS1-CAR cells at an E/T ratio of 5:1for 24 h. Data shown are representative of one out of three patientsamples with similar results.

FIGS. 19A to 19D show CS1-CAR NK cells suppress in vivo growth oforthotopic human MM cells and prolong the survival of MM-bearing mice.FIG. 19A (left) is an image showing massive infiltration of human IM9cells, detected by Hematoxylin-Eosin (H&E) staining, in the lumbarvertebrae bone lesions of one representative mouse displaying hind legparalyses after i.v. injected with IM9 cells. FIG. 19A (right) showsimmunohistochemical staining of mouse lumbar vertebrae bone lesions withanti-human CD138 mAb. FIG. 19B shows dorsal bioluminescence imaging ofmice bearing IM9 tumors. NSG mice were inoculated with 5×10⁵luciferase-expressing IM9 cells via a tail vein injection (day 0). Sevendays after inoculation, mice were treated with mock-transduced NK-92cells (NK-92-EV), CS1-CAR transduced NK-92 cells (NK-92-CS1-CAR) orphosphate-buffered saline (a negative control). FIG. 19C is a bar graphshowing quantification summary of units of photons per second per mousefrom FIG. 19B. * indicates P<0.05; ** denotes P<0.01. FIG. 19D showsKaplan-Meier survival curves of IM9-bearing mice treated withNK-92-CS1-CAR cells compared with the mice treated with NK-92-EVcells. * indicates P<0.05.

FIG. 20 shows that introduction of CS1 CAR does not lead to substantialapoptosis in NK cell lines. Mock- or CS1-CAR-transduced NK92 or NKLcells were stained with 7AAD and Annexin V-V450, followed by a flowcytometric analysis. NK-92-EV and NKL-EV indicate empty vector (EV)control-transduced NK-92 and NKL cells, respectively. NK-92-CS1-CAR andNKL-CS1-CAR indicate transduction of NK-92 and NKL cells, respectively,with a CS1-CAR construct.

DETAILED DESCRIPTION

Disclosed herein are chimeric antigen receptors (CAR) that canspecifically recognize tumor-associated antigens (TAA) on Multiplemyeloma (MM) cells. Also disclosed are immune effector cells, such as Tcells or Natural Killer (NK) cells, that are engineered to express theseCARs. Therefore, also disclosed are methods for providing an anti-tumorimmunity in a subject with MM that involves adoptive transfer of thedisclosed immune effector cells engineered to express the disclosedCS1-specific CARs.

CS1-Specific Chimeric Antigen Receptors (CAR)

CARs generally incorporate an antigen recognition domain from thesingle-chain variable fragments (scFv) of a monoclonal antibody (mAb)with transmembrane signaling motifs involved in lymphocyte activation(Sadelain M, et al. Nat Rev Cancer 2003 3:35-45). The cell surfaceglycoprotein CS1 is highly and ubiquitously expressed on the surface ofmyeloma cells (Hsi E D, et al. Clin Cancer Res 2008 14:2775-84). CS1 isexpressed at very low levels in the majority of immune effector cells,including natural killer (NK) cells, some subsets of T cells, and normalB cells, and is almost undetectable on myeloid cells (Hsi E D, et al.Clin Cancer Res 2008 14:2775-84). Notably, CS1 is negligibly expressedin human hematopoietic stem cells (Hsi E D, et al. Clin Cancer Res 200814:2775-84), which can be used for stem cell transplantation to treathematologic malignancies, including MM. The functions of CS1 in MMremain incompletely understood, and it has been documented that CS1 mayplay a role in myeloma cell adhesion, clonogenic growth, andtumorigenicity (Benson D M Jr, et al. J Clin Oncol 2012 30:2013-5; Tai YT, et al. Blood 2009 113:4309-18). Targeting CS1 with the humanized mAbelotuzumab has been demonstrated to be safe in the clinic (Benson D MJr, et al. J Clin Oncol 2012 30:2013-5; Tai Y T, et al. Blood 2009113:4309-18). Preclinical studies show that this antibody inhibitsmyeloma cell adhesion to bone marrow stromal cells, induces NKcell-mediated antibody-dependent cellular cytotoxicity, and eradicatesthe xenograft tumors initiated by human myeloma cells in immunodeficientmice (Benson D M Jr, et al. J Clin Oncol 2012 30:2013-5; Tai Y T, et al.Blood 2009 113:4309-18; Tai Y T, et al. Blood 2008 112:1329-37).Therefore, disclosed herein is a CS1-specific chimeric antigen receptor(CAR) that can be that can be expressed in immune effector cells toenhance antitumor activity against human multiple myeloma.

The disclosed CAR is generally made up of three domains: an ectodomain,a transmembrane domain, and an endodomain. The ectodomain comprises theCS1-binding region and is responsible for antigen recognition. It alsogenerally contains a signal peptide (SP) so that the CAR can beglycosylated and anchored in the cell membrane of the immune effectorcell. The transmembrane domain (TD), is as its name suggests, connectsthe ectodomain to the endodomain and resides within the cell membranewhen expressed by a cell. The endodomain is the business end of the CARthat transmits an activation signal to the immune effector cell afterantigen recognition. For example, the endodomain can contain anintracellular signaling domain (ISD) and optionally a co-stimulatorysignaling region (CSR).

In some embodiments, the disclosed CAR is defined by the formula:SP-CS1-HG-TM-CSR-ISD; orSP-CS1-HG-TM-ISD-CSR

wherein “SP” represents a signal peptide,

wherein “CS1” represents a CS1-binding region,

wherein “HG” represents an optional hinge domain,

wherein “TM” represents a transmembrane domain,

wherein “CSR” represents a co-stimulatory signaling region,

wherein “ISD” represents an intracellular signaling domain, and

wherein “-” represents a bivalent linker.

The antigen recognition domain of the disclosed CAR is usually an scFv.There are however many alternatives. An antigen recognition domain fromnative T-cell receptor (TCR) alpha and beta single chains have beendescribed, as have simple ectodomains (e.g. CD4 ectodomain to recognizeHIV infected cells) and more exotic recognition components such as alinked cytokine (which leads to recognition of cells bearing thecytokine receptor). In fact almost anything that binds a given targetwith high affinity can be used as an antigen recognition region.

The endodomain is the business end of the CAR that after antigenrecognition (i.e., CS1) transmits a signal to the immune effector cell,activating at least one of the normal effector functions of the immuneeffector cell. Effector function of a T cell, for example, may becytolytic activity or helper activity including the secretion ofcytokines. Therefore, the endodomain may comprise the “intracellularsignaling domain” of a T cell receptor (TCR) and optional co-receptors.While usually the entire intracellular signaling domain can be employed,in many cases it is not necessary to use the entire chain. To the extentthat a truncated portion of the intracellular signaling domain is used,such truncated portion may be used in place of the intact chain as longas it transduces the effector function signal.

Cytoplasmic signaling sequences that regulate primary activation of theTCR complex that act in a stimulatory manner may contain signalingmotifs which are known as immunoreceptor tyrosine-based activationmotifs (ITAMs). Examples of ITAM containing cytoplasmic signalingsequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.However, in preferred embodiments, the intracellular signaling domain isderived from CD3 zeta (CD3ζ).

T-cell surface glycoprotein CD3 zeta (CD3ζ) chain, also known as T-cellreceptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is aprotein that in humans is encoded by the CD247 gene.

First-generation CARs typically had the intracellular domain from theCD3ζ chain, which is the primary transmitter of signals from endogenousTCRs. Second-generation CARs add intracellular signaling domains fromvarious costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to theendodomain of the CAR to provide additional signals to the T cell.Preclinical studies have indicated that the second generation of CARdesigns improves the antitumor activity of T cells. More recent,third-generation CARs combine multiple signaling domains to furtheraugment potency. T cells grafted with these CARs have demonstratedimproved expansion, activation, persistence, and tumor-eradicatingefficiency independent of costimulatory receptor/ligand interaction(Imai C, et al. Leukemia 2004 18:676-84; Maher J, et al. Nat Biotechnol2002 20:70-5).

For example, the endodomain of the CAR can be designed to comprise theCD3ζ signaling domain by itself or combined with any other desiredcytoplasmic domain(s) useful in the context of the CAR of the invention.For example, the cytoplasmic domain of the CAR can comprise a CD3 zetachain portion and a costimulatory signaling region. The costimulatorysignaling region refers to a portion of the CAR comprising theintracellular domain of a costimulatory molecule. A costimulatorymolecule is a cell surface molecule other than an antigen receptor ortheir ligands that is required for an efficient response of lymphocytesto an antigen. Examples of such molecules include CD27, CD28, 4-1BB(CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand thatspecifically binds with CD83, and the like. Thus, while the CAR isexemplified primarily with CD28 as the co-stimulatory signaling element,other costimulatory elements can be used alone or in combination withother co-stimulatory signaling elements.

In some embodiments, the CAR comprises a hinge sequence. A hingesequence is a short sequence of amino acids that facilitates antibodyflexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99(2004)). The hinge sequence may be positioned between the antigenrecognition moiety (e.g., an anti-CS1 scFv) and the transmembranedomain. The hinge sequence can be any suitable sequence derived orobtained from any suitable molecule. In some embodiments, for example,the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. For example, thetransmembrane region may be derived from (i.e. comprise at least thetransmembrane region(s) of) the alpha, beta or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33,CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively thetransmembrane domain may be synthetic, in which case it will comprisepredominantly hydrophobic residues such as leucine and valine. In somecases, a triplet of phenylalanine, tryptophan and valine will be foundat each end of a synthetic transmembrane domain. A short oligo- orpolypeptide linker, such as between 2 and 10 amino acids in length, mayform the linkage between the transmembrane domain and the endoplasmicdomain of the CAR.

The bivalent linker can be any molecule suitable to link a compound ornucleic acid to a polynucleotide sequence. Methods and compositions forconjugating biomolecules, such as polynucleotides, are disclosed in G.T. Hermanon, Bioconjugate Techniques (2^(nd) ed.), Academic Press(2008), which is incorporated by reference in its entirety for theteaching of these techniques. In some cases, the bivalent linkercomprises one or more amino acids. However, it can also comprise apeptide bond directly linking the disclosed domains.

In some embodiments, the disclosed CS1-specific CAR comprises one ormore of the SP, CS1, HG, TM, CSR, ISD, and/or linker components setforth in Table 1, or variants thereof having at least 65%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity to the sequences set forth in Table 1.

TABLE 1 Exemplary sequences for the different CAR CS1 componentsFunctional Amino Acid Sequence/ domains SEQ ID # Nucleic Acid SequenceSP SEQ ID NO: 1 MGWSSIILFLVATATGVH SEQ ID NO: 2ATGGGATGGAGCTCTATCATCCTCTTCTTGGTAGCAA CAGCTACAGGTGTCCAC CD8α SPSEQ ID NO: 3 MALPVTALLLPLALLLHAARP Alternative SP SEQ ID NO: 4METDTLLLWVLLLWVPGSTG Hinge domain SEQ ID NO: 5 LEPKSCDKTHTCPPCPSEQ ID NO: 6 CTCGAGCCCAAATCTTGTGACAAAACTCACACATGC CCACCGTGCCCG CD8α TMSEQ ID NO: 7 IYIWAPLAGTCGVLLLSLVITLYC 41BB TM SEQ ID NO: 8IISFFLALTSTALLFLLFFLTLRFSVV CD28 TM SEQ ID NO: 9FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 10TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTT CTGGGTG 41BB CSR SEQ ID NO: 11KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCEL CD28 CSR SEQ ID NO: 12RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA AYRS SEQ ID NO: 13AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTC GCAGCCTATCGCTCC CDζ ISDSEQ ID NO: 14 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR SEQ ID NO: 15AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACA ATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGC AAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCTAA LinkerSEQ ID NO: 16 GGGGSGGGGSGGGGS Luc90 CS1 ScFv SEQ ID NO: 17SQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSGGGGSGGGGSGGGGSDIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYY CQQHYSTPLTFGAGTKLELKSEQ ID NO: 18 TCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCGGGGTACTCCTTCACCACCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCTACTATGATTGCGACGAGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCCGGCGGTGGCGGTTCTGACATTGTGATGACCCAGTCTCAGAAATCCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTTATTACTGGTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAATTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAATGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCTCTCACTTTCGGTGCTGGGACCAAGCTGGAGCTGAAA Luc90 light chain SEQ ID NO: 19DIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQ variable regionQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTIS NVQAEDLAVYYCQQHYSTPLTFGAGTKLELKLuc63 heavy chain SEQ ID NO: 20 EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVvariable region RQAPGKGLEWIGEINPDSSTINYTPSLKDKFITSRDNAKNTLYLQMSKVRSEDTALYYCARPDGNYWYFDVWGAGT TVTVSS Luc63 light chainSEQ ID NO: 21 DIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQ variable regionQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTIS NVQSEDLADYFCQQYSSYPYTFGGGTKLEIKLuc34 heavy chain SEQ ID NO: 22 QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWvariable region VKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAY WGQGTLVTVSA Luc34 light chainSEQ ID NO: 23 DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQ variable regionKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSL QTEDVATYYCQQYWSTPWTFGGGTKLEIKLucX1 heavy SEQ ID NO: 24 QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVchain variable KQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADK regionSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWG QGTSVTVSS LucX1 light chainSEQ ID NO: 25 ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQ variable regionKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENM LSEDVADYYCLQSDNLPLTFGGGTKLEIKLucX2 heavy SEQ ID NO: 26 QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVchain variable KQRPGQGLEWTGRTYPGDGDTKYNGKFKGKATLTADK regionSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWG QGTSVTVS LucX2 light chainSEQ ID NO: 27 DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQ variable regionQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISS VQAEDLAVYYCQQHYSTPPYTFGGGTKLEIK

Therefore, in some embodiments, the disclosed CS1-specific CAR comprisesthe amino acid sequence SEQ ID NO:1 (shown below), or a variant thereofhaving at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:28.

CS1-CD28-CD3Z construct:

(SEQ ID NO: 28) MGWSSIILFLVATATGVHSQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSGGGGSGGGGSGGGGSDIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKWYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYYCQQHYSTPLTFGAGTKLELKLEPKSCDKTHTCPPCPDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR.

Nucleic Acids and Vectors

Also disclosed are polynucleotides and polynucleotide vectors encodingthe disclosed CS1-specific CARs that allow expression of theCS1-specific CARs in the disclosed immune effector cells.

For example, in some embodiments, the disclosed CS1-specific CAR areencoded by the nucleic acid sequence SEQ ID NO:28 (shown below), or avariant thereof having at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ IDNO:29.

PCDH-CS1-scFv-myc tag-CD28-CD3zeta (PCDH-CS1-CAR) construct:

(SEQ ID NO: 29) ATGGGATGGAGCTCTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCGGGGTACTCCTTCACCACCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGCATGATTCATCCTTCCGATAGTGAAACTAGGTTAAATCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCTACTATGATTGCGACGAGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCGGCGGTGGCGGTTCTGGTGGCGGTGGCTCCGGCGGTGGCGGTTCTGACATTGTGATGACCCAGTCTCAGAAATCCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTTATTACTGGTGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAATTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAATGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCTCTCACTTTCGGTGCTGGGACCAAGCTGGAGCTGAAACTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCGGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCACCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA.

Nucleic acid sequences encoding the disclosed CARs, and regions thereof,can be obtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

Expression of nucleic acids encoding CARs is typically achieved byoperably linking a nucleic acid encoding the CAR polypeptide to apromoter, and incorporating the construct into an expression vector.Typical cloning vectors contain transcription and translationterminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence.

The disclosed nucleic acid can be cloned into a number of types ofvectors. For example, the nucleic acid can be cloned into a vectorincluding, but not limited to a plasmid, a phagemid, a phage derivative,an animal virus, and a cosmid. Vectors of particular interest includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers. In someembodiments, the polynucleotide vectors are lentiviral or retroviralvectors.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α(EF-1α). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, MND (myeloproliferative sarcoma virus) promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the hemoglobin promoter,and the creatine kinase promoter. The promoter can alternatively be aninducible promoter. Examples of inducible promoters include, but are notlimited to a metallothionine promoter, a glucocorticoid promoter, aprogesterone promoter, and a tetracycline promoter.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene. Suitableexpression systems are well known and may be prepared using knowntechniques or obtained commercially. In general, the construct with theminimal 5′ flanking region showing the highest level of expression ofreporter gene is identified as the promoter. Such promoter regions maybe linked to a reporter gene and used to evaluate agents for the abilityto modulate promoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for usecan be obtained from commercial sources. For example, dimyristylphosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.;dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories(Plainview, N.Y.); cholesterol (“Choi”) can be obtained fromCalbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and otherlipids may be obtained from Avanti Polar Lipids, Inc, (Birmingham,Ala.).

Immune Effector Cells

Also disclosed are immune effector cells that are engineered to expressthe disclosed CARs. These cells are preferably obtained from the subjectto be treated (i.e. are autologous). However, in some embodiments,immune effector cell lines or donor effector cells (allogeneic) areused. Immune effector cells can be obtained from a number of sources,including peripheral blood mononuclear cells, bone marrow, lymph nodetissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. Immune effectorcells can be obtained from blood collected from a subject using anynumber of techniques known to the skilled artisan, such as Ficoll™separation. For example, cells from the circulating blood of anindividual may be obtained by apheresis. In some embodiments, immuneeffector cells are isolated from peripheral blood lymphocytes by lysingthe red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalclutriation. A specific subpopulation of immune effector cells can befurther isolated by positive or negative selection techniques. Forexample, immune effector cells can be isolated using a combination ofantibodies directed to surface markers unique to the positively selectedcells, e.g., by incubation with antibody-conjugated beads for a timeperiod sufficient for positive selection of the desired immune effectorcells. Alternatively, enrichment of immune effector cells population canbe accomplished by negative selection using a combination of antibodiesdirected to surface markers unique to the negatively selected cells.

In some embodiments, the immune effector cells comprise any leukocyteinvolved in defending the body against infectious disease and foreignmaterials. For example, the immune effector cells can compriselymphocytes, monocytes, macrophages, dentritic cells, mast cells,neutrophils, basophils, eosinophils, or any combinations thereof. Forexample, the immune effector cells can comprise T lymphocytes.

T cells or T lymphocytes can be distinguished from other lymphocytes,such as B cells and natural killer cells (NK cells), by the presence ofa T-cell receptor (TCR) on the cell surface. They are called T cellsbecause they mature in the thymus (although some also mature in thetonsils). There are several subsets of T cells, each with a distinctfunction.

T helper cells (T_(H) cells) assist other white blood cells inimmunologic processes, including maturation of B cells into plasma cellsand memory B cells, and activation of cytotoxic T cells and macrophages.These cells are also known as CD4+ T cells because they express the CD4glycoprotein on their surface. Helper T cells become activated when theyare presented with peptide antigens by MHC class II molecules, which areexpressed on the surface of antigen-presenting cells (APCs). Onceactivated, they divide rapidly and secrete small proteins calledcytokines that regulate or assist in the active immune response. Thesecells can differentiate into one of several subtypes, including T_(H)1,T_(H)2, T_(H)3, T_(H)17, T_(H)9, or T_(FH), which secrete differentcytokines to facilitate a different type of immune response.

Cytotoxic T cells (T_(C) cells, or CTLs) destroy virally infected cellsand tumor cells, and are also implicated in transplant rejection. Thesecells are also known as CD8⁺ T cells since they express the CD8glycoprotein at their surface. These cells recognize their targets bybinding to antigen associated with MHC class I molecules, which arepresent on the surface of all nucleated cells. Through IL-10, adenosineand other molecules secreted by regulatory T cells, the CD8+ cells canbe inactivated to an anergic state, which prevents autoimmune diseases.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory cells may be either CD4⁺ or CD8⁺. Memory T cells typicallyexpress the cell surface protein CD45RO.

Regulatory T cells (T_(reg) cells), formerly known as suppressor Tcells, are crucial for the maintenance of immunological tolerance. Theirmajor role is to shut down T cell-mediated immunity toward the end of animmune reaction and to suppress auto-reactive T cells that escaped theprocess of negative selection in the thymus. Two major classes of CD4⁺T_(reg) cells have been described—naturally occurring T_(reg) cells andadaptive T_(reg) cells.

Natural killer T (NKT) cells (not to be confused with natural killer(NK) cells) bridge the adaptive immune system with the innate immunesystem. Unlike conventional T cells that recognize peptide antigenspresented by major histocompatibility complex (MHC) molecules, NKT cellsrecognize glycolipid antigen presented by a molecule called CD1d.

In some embodiments, the T cells comprise a mixture of CD4+ cells. Inother embodiments, the T cells are enriched for one or more subsetsbased on cell surface expression. For example, in some cases, the Tcomprise are cytotoxic CD8⁺ T lymphocytes. In some embodiments, the Tcells comprise γδ T cells, which possess a distinct T-cell receptor(TCR) having one γ chain and one δ chain instead of α and β chains.

Natural-killer (NK) cells are CD56⁺CD3⁻ large granular lymphocytes thatcan kill virally infected and transformed cells, and constitute acritical cellular subset of the innate immune system (Godfrey J, et al.Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8⁺ T lymphocytes,NK cells launch cytotoxicity against tumor cells without the requirementfor prior sensitization, and can also eradicate MHC-I-negative cells(Narni-Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells aresafer effector cells, as they may avoid the potentially lethalcomplications of cytokine storms (Morgan R A, et al. Mol Ther 201018:843-851), tumor lysis syndrome (Porter D L, et al. N Engl J Med 2011365:725-733), and on-target, off-tumor effects. Although NK cells have awell-known role as killers of cancer cells, and NK cell impairment hasbeen extensively documented as crucial for progression of MM (Godfrey J,et al. Leuk Lymphoma 2012 53:1666-1676; Fauriat C, et al. Leukemia 200620:732-733), the means by which one might enhance NK cell-mediatedanti-MM activity has been largely unexplored prior to the disclosedCARs.

Therapeutic Methods

Immune effector cells expressing the disclosed CARs can elicit ananti-tumor immune response against MM cells. The anti-tumor immuneresponse elicited by the disclosed CAR-modified immune effector cellsmay be an active or a passive immune response. In addition, theCAR-mediated immune response may be part of an adoptive immunotherapyapproach in which CAR-modified immune effector cells induce an immuneresponse specific to CS1.

Adoptive transfer of immune effector cells expressing chimeric antigenreceptors is a promising anti-cancer therapeutic. Following thecollection of a patient's immune effector cells, the cells may begenetically engineered to express the disclosed CS1-specific CARs, theninfused back into the patient.

The disclosed CAR-modified immune effector cells may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2, IL-15, or othercytokines or cell populations. Briefly, pharmaceutical compositions maycomprise a target cell population as described herein, in combinationwith one or more pharmaceutically or physiologically acceptablecarriers, diluents or excipients. Such compositions may comprise bufferssuch as neutral buffered saline, phosphate buffered saline and the like;carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;proteins; polypeptides or amino acids such as glycine; antioxidants;chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions for use in the disclosedmethods are in some embodiments formulated for intravenousadministration. Pharmaceutical compositions may be administered in anymanner appropriate treat MM. The quantity and frequency ofadministration will be determined by such factors as the condition ofthe patient, and the severity of the patient's disease, althoughappropriate dosages may be determined by clinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “an tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, such as 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently re-draw blood (or have anapheresis performed), activate T cells therefrom according to thedisclosed methods, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Using this multiple blood draw/multiplereinfusion protocol may serve to select out certain populations of Tcells.

The administration of the disclosed compositions may be carried out inany convenient manner, including by injection, transfusion, orimplantation. The compositions described herein may be administered to apatient subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In some embodiments, the disclosed compositions areadministered to a patient by intradermal or subcutaneous injection. Insome embodiments, the disclosed compositions are administered by i.v.injection. The compositions may also be injected directly into a tumor,lymph node, or site of infection.

In certain embodiments, the disclosed CAR-modified immune effector cellsare administered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to thalidomide, dexamethasone,bortezomib, and lenalidomide. In further embodiments, the CAR-modifiedimmune effector cells may be used in combination with chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAM PATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation. Insome embodiments, the CAR-modified immune effector cells areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in someembodiments, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

Definitions

The term “amino acid sequence” refers to a list of abbreviations,letters, characters or words representing amino acid residues. The aminoacid abbreviations used herein are conventional one letter codes for theamino acids and are expressed as follows: A, alanine; B, asparagine oraspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamicacid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K,lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q,glutamine; R, arginine; S, serine; T, threonine; V, valine; W,tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

The term “antibody” refers to natural or synthetic antibodies thatselectively bind a target antigen. The term includes polyclonal andmonoclonal antibodies. In addition to intact immunoglobulin molecules,also included in the term “antibodies” are fragments or polymers ofthose immunoglobulin molecules, and human or humanized versions ofimmunoglobulin molecules that selectively bind the target antigen.

The term “aptamer” refers to oligonucleic acid or peptide molecules thatbind to a specific target molecule. These molecules are generallyselected from a random sequence pool. The selected aptamers are capableof adapting unique tertiary structures and recognizing target moleculeswith high affinity and specificity. A “nucleic acid aptamer” is a DNA orRNA oligonucleic acid that binds to a target molecule via itsconformation, and thereby inhibits or suppresses functions of suchmolecule. A nucleic acid aptamer may be constituted by DNA, RNA, or acombination thereof. A “peptide aptamer” is a combinatorial proteinmolecule with a variable peptide sequence inserted within a constantscaffold protein. Identification of peptide aptamers is typicallyperformed under stringent yeast dihybrid conditions, which enhances theprobability for the selected peptide aptamers to be stably expressed andcorrectly folded in an intracellular context.

The term “carrier” means a compound, composition, substance, orstructure that, when in combination with a compound or composition, aidsor facilitates preparation, storage, administration, delivery,effectiveness, selectivity, or any other feature of the compound orcomposition for its intended use or purpose. For example, a carrier canbe selected to minimize any degradation of the active ingredient and tominimize any adverse side effects in the subject.

The term “chimeric molecule” refers to a single molecule created byjoining two or more molecules that exist separately in their nativestate. The single, chimeric molecule has the desired functionality ofall of its constituent molecules. One type of chimeric molecules is afusion protein.

The term “fusion protein” refers to a polypeptide formed by the joiningof two or more polypeptides through a peptide bond formed between theamino terminus of one polypeptide and the carboxyl terminus of anotherpolypeptide. The fusion protein can be formed by the chemical couplingof the constituent polypeptides or it can be expressed as a singlepolypeptide from nucleic acid sequence encoding the single contiguousfusion protein. A single chain fusion protein is a fusion protein havinga single contiguous polypeptide backbone. Fusion proteins can beprepared using conventional techniques in molecular biology to join thetwo genes in frame into a single nucleic acid, and then expressing thenucleic acid in an appropriate host cell under conditions in which thefusion protein is produced.

The term “identity” refers to sequence identity between two nucleic acidmolecules or polypeptides. Identity can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base, then the molecules are identical at that position. A degreeof similarity or identity between nucleic acid or amino acid sequencesis a function of the number of identical or matching nucleotides atpositions shared by the nucleic acid sequences. Various alignmentalgorithms and/or programs may be used to calculate the identity betweentwo sequences, including FASTA, or BLAST which are available as a partof the GCG sequence analysis package (University of Wisconsin, Madison,Wis.), and can be used with, e.g., default setting. For example,polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity tospecific polypeptides described herein and preferably exhibitingsubstantially the same functions, as well as polynucleotide encodingsuch polypeptides, are contemplated. Unless otherwise indicated asimilarity score will be based on use of BLOSUM62. When BLASTP is used,the percent similarity is based on the BLASTP positives score and thepercent sequence identity is based on the BLASTP identities score.BLASTP “Identities” shows the number and fraction of total residues inthe high scoring sequence pairs which are identical; and BLASTP“Positives” shows the number and fraction of residues for which thealignment scores have positive values and which are similar to eachother. Amino acid sequences having these degrees of identity orsimilarity or any intermediate degree of identity of similarity to theamino acid sequences disclosed herein are contemplated and encompassedby this disclosure. The polynucleotide sequences of similar polypeptidesare deduced using the genetic code and may be obtained by conventionalmeans, in particular by reverse translating its amino acid sequenceusing the genetic code.

The term “nucleic acid” refers to a natural or synthetic moleculecomprising a single nucleotide or two or more nucleotides linked by aphosphate group at the 3′ position of one nucleotide to the 5′ end ofanother nucleotide. The nucleic acid is not limited by length, and thusthe nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleicacid (RNA).

The term “operably linked to” refers to the functional relationship of anucleic acid with another nucleic acid sequence. Promoters, enhancers,transcriptional and translational stop sites, and other signal sequencesare examples of nucleic acid sequences operably linked to othersequences. For example, operable linkage of DNA to a transcriptionalcontrol element refers to the physical and functional relationshipbetween the DNA and promoter such that the transcription of such DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes, binds to and transcribes the DNA.

The terms “peptide,” “protein,” and “polypeptide” are usedinterchangeably to refer to a natural or synthetic molecule comprisingtwo or more amino acids linked by the carboxyl group of one amino acidto the alpha amino group of another.

The term “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

The term “protein domain” refers to a portion of a protein, portions ofa protein, or an entire protein showing structural integrity; thisdetermination may be based on amino acid composition of a portion of aprotein, portions of a protein, or the entire protein.

A “spacer” as used herein refers to a peptide that joins the proteinscomprising a fusion protein. Generally a spacer has no specificbiological activity other than to join the proteins or to preserve someminimum distance or other spatial relationship between them. However,the constituent amino acids of a spacer may be selected to influencesome property of the molecule such as the folding, net charge, orhydrophobicity of the molecule.

The term “specifically binds”, as used herein, when referring to apolypeptide (including antibodies) or receptor, refers to a bindingreaction which is determinative of the presence of the protein orpolypeptide or receptor in a heterogeneous population of proteins andother biologics. Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody), a specified ligand or antibody“specifically binds” to its particular “target” (e.g. an antibodyspecifically binds to an endothelial antigen) when it does not bind in asignificant amount to other proteins present in the sample or to otherproteins to which the ligand or antibody may come in contact in anorganism. Generally, a first molecule that “specifically binds” a secondmolecule has an affinity constant (Ka) greater than about 10⁵ M⁻¹ (e.g.,10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, and 10¹² M⁻¹ ormore) with that second molecule.

The term “specifically deliver” as used herein refers to thepreferential association of a molecule with a cell or tissue bearing aparticular target molecule or marker and not to cells or tissues lackingthat target molecule. It is, of course, recognized that a certain degreeof non-specific interaction may occur between a molecule and anon-target cell or tissue. Nevertheless, specific delivery, may bedistinguished as mediated through specific recognition of the targetmolecule. Typically specific delivery results in a much strongerassociation between the delivered molecule and cells bearing the targetmolecule than between the delivered molecule and cells lacking thetarget molecule.

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient. The term “patient” refers to a subject under the treatment of aclinician, e.g., physician.

The term “therapeutically effective” refers to the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination.

The terms “transformation” and “transfection” mean the introduction of anucleic acid, e.g., an expression vector, into a recipient cellincluding introduction of a nucleic acid to the chromosomal DNA of saidcell.

The term “treatment” refers to the medical management of a patient withthe intent to cure, ameliorate, stabilize, or prevent a disease,pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The term “variant” refers to an amino acid or peptide sequence havingconservative amino acid substitutions, non-conservative amino acidsubsitutions (i.e. a degenerate variant), substitutions within thewobble position of each codon (i.e. DNA and RNA) encoding an amino acid,amino acids added to the C-terminus of a peptide, or a peptide having60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to areference sequence.

The term “vector” refers to a nucleic acid sequence capable oftransporting into a cell another nucleic acid to which the vectorsequence has been linked. The term “expression vector” includes anyvector, (e.g., a plasmid, cosmid or phage chromosome) containing a geneconstruct in a form suitable for expression by a cell (e.g., linked to atranscriptional control element).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES Example 1: Genetic Modification of T Cells Redirected TowardsCS1 Enhances Eradication of Myeloma Cells

In the present study, T cells were manipulated to express a CS1-specificCAR incorporating CD28-CD3ζ signaling moieties, demonstrating thatCS1-specific CAR T cells mediated enhanced cytokine release andcytotoxicity in response to CS1-expressing myeloma cells, which occurredin a CS1-dependent manner. Moreover, in orthotopic MM xenograft mousemodels, CS1-redirected T cells efficiently eradicated human myelomacells and significantly prolonged mouse survival. Together, these datasuggest that adoptive therapy with T cells armed with a CS1-specific CARrepresents a promising strategy against relapsed MM.

Materials and Methods

Cell Culture

Human multiple myeloma cell lines IM9, NCI-H929, MM.1S and RPMI-8226were obtained from the American Type Culture Collection (ATCC), andmaintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetalbovine serum (FBS) (Invitrogen). 293T and phoenix packaging cells werecultivated in DMEM medium (Invitrogen) with 10% FBS. Human peripheralblood mononuclear cells (PBMCs) from healthy donors and multiple myelomapatients were isolated by Ficoll-Paque Plus (GE Healthcare Bio-Sciences,Pittsburgh, Pa.) density gradient centrifugation, and monocytes weredepleted by plastic adherence. Primary CD138⁺ myeloma cells werepositively selected from bone marrow aspirates of patients using humananti-CD138 MicroBeads and magnet-assisted cell sorting (MACS, MiltenyiBiotech), according to the manufacturer's instructions. Informed consentwas obtained from myeloma patients according to a protocol approved byThe Ohio State University Institutional Review Board.

Mice

Six- to 8-week-old male NOD-scid IL-2Rgamma null (NSG) mice wereobtained from The Jackson Laboratory. Mice were monitored frequently forMM disease progression, and sacrificed when they became moribund withthe symptoms of hind limb paralysis, lethargy, or obvious weight loss.All animal work was approved by The Ohio State University Animal Careand Use Committee.

Generation of the CS1-Specific CAR Retroviral Construct

The anti-CS1 scFv was derived from the hybridoma cell line Luc90. Thecoding domain sequences for variable regions of heavy (V_(H)) and light(V_(L)) chains were amplified separately and recombined using a linkerby overlapping PCR reaction. The V_(H)-linker-V_(L) fragment wasincorporated in frame with the CD28-CD3ζ portion. The entireanti-CS1-scFv-CD28-CD3 fragment was then ligated into a retroviralvector designated Pinco (Yu J, et al. Immunity 2006 24:575-90; BecknellB, et al. J Immunol Methods 2005 296:115-23) to generate a Pinco-CS1-CARconstruct.

Retroviral Transduction of T Lymphocytes

Retroviral supernatants were collected from phoenix packaging cellstransiently transfected with the Pinco- or Pinco-CS1-CAR construct for48 h, as described previously (Becknell B, et al. J Immunol Methods.2005 296(1-2):115-123; Yu J, et al. Immunity. 2006 24(5):575-590). PBMCswere cultured in RPMI 1640 medium with 10% FBS and stimulated with HumanT-Activator CD3/CD28 Dynabeads (Invitrogen) and 150 IU/mL humanrecombinant interleukin-2 (IL-2, Hoffman-La Roche Inc.) for 2 days. Thencells were resuspended in infectious supernatants and applied intoRETRONECTIN (Clontech Laboratories)-coated non-tissue culture-treated6-well plates according to the manufacturer's protocol. The infectionprocess was repeated once on the second day. Then cells were transferredinto tissue-culture-treated flasks and maintained in the presence of 150IU/mL IL-2. Transduced T cells were purified using a FACSARIA II cellsorter (BD Biosciences) based on expression of a GFP marker on the cellsurface encoded by the Pinco vector.

Flow Cytometry Analysis

For detection of CS1-CAR expression on the cell surface, transduced Tcells were washed with PBS containing 4% bovine serum albumin, andincubated with biotin-labeled goat anti-mouse (Fab)2 polyclonal antibodyor normal polyclonal goat immunoglobulin G (IgG) antibody (JacksonImmunoResearch) as an isotype control. Then cells were stained withallophycocyanin (APC)-conjugated streptavidin (Jackson ImmunoResearch)and anti-CD3 antibody conjugated to V450 (BD Biosciences). To determinethe expression of CS1 on the surface of myeloma cells, the cells werestained with phycoerythrin (PE)-conjugated mouse anti-CS1 mAb(eBiosciences) and APC-conjugated mouse anti-CD138 mAb (MiltenyiBiotec). Antibody staining was monitored with a BD LSRII flow cytometer.Data analysis was carried out using FLOWJO software (Tree Star Inc.)

Immunoblotting

Cells were lysed in laemmli buffer. Lysates were separated by SDS-PAGEgel and transferred to nitrocellulose membrane (Millipore). The membranewas probed with mouse anti-human CD3ζ mAb (BD Pharmingen) and then witha horseradish peroxidase-conjugated goat anti-mouse IgG antibody.Antibody binding was revealed by using an enhanced chemiluminescencereagent (GE Healthcare Biosciences).

Generation of RPMI-8226 Cells Stably Expressing CS1

Full length human CS1 coding sequence was PCR-amplified from the IM9cDNA, and inserted into a lentiviral vector designatedPCDH-CMV-MCS-EF1-copGFP (PCDH, System Biosciences), yielding PCDH-CS1.To produce lentivirus, 293T cells were co-transfected with the PCDH-CS1plasmid or a PCDH empty vector plasmid plus the packaging plasmidspCMV-VSVG and pCMV-dr9 using calcium phosphate transfection reagent(Promega). Then, the lentiviral supernatants were harvested and used toinfect RPMI-8226 cells using a previously published protocol (BecknellB, et al. J Immunol Methods. 2005 296(1-2):115-123; Yu J, et al.Immunity. 2006 24(5):575-590).

Cytotoxicity Assay

A standard 4-hour ⁵¹Cr release assay was performed as describedpreviously (Yu J, et al. Blood 2010 115:274-81). Briefly, target cellswere labeled with ⁵¹Cr and co-cultured with T cells at variouseffector/target ratios (E/T) in the wells of 96-well V-bottom plate at37° C. for 4 hours. Supernatants were harvested and transferred intoscintillation vials containing a liquid scintillation cocktail (FisherScientific), and the release of ⁵¹Cr was measured on TOPCOUNT counter(Canberra Packard). Target cells incubated in complete medium or 1% SDSwere used to determine spontaneous or maximal ⁵¹Cr release. Thepercentage of specific lysis was calculated using the standard formula:100×(cpm experimental release−cpm spontaneous release)/(cpm maximalrelease−cpm spontaneous release).

Cytokine Release Assays

Target cells were co-cultured with an equal number of effector cells in96-well V-bottom plates at 37° C. for 24 hours. Cell-free supernatantswere harvested and assessed for IFN-g and interleukin (IL)-2 secretionby ELISA using corresponding ELISA kits from R&D system according to themanufacturer's protocol.

CD107a Degranulation Assay

CD107a assay was performed as described previously with somemodification (He S, et al. Blood 2013 121:4663-71). Briefly, MM targetcells (2.5×10⁵) were co-cultured with an equal number of effector cellsin 0.2 mL per well in 96-well V-bottom plates. Control cells are eithermock- or CS1-CAR-transduced T cells incubated without target cells.Anti-CD107a or IgG1 isotype antibody conjugated to APC (BD Biosciences)together with 1 mL monensin (BD Biosciences) was added and incubated at37° C. for 4 hours. Cells were further stained with PE-conjugated CD69and V450-conjugated CD3 antibodies, and analyzed using a LSRII flowcytometer (BD Biosciences).

Intracellular Staining of Granzyme B and Perforin

Mock- or CS1-CAR-transduced T cells were washed and stained withV450-conjugated anti-human CD3 mAb. Subsequently, cells were fixed andpermeabilized using the Cytofix/Cytoperm Kit (BD Biosciences), labeledwith APC-conjugated anti-granzyme B (Invitrogen), APC-conjugatedanti-perforin antibody (eBiosciences) or a mouse APC-conjugated isotypeantibody, and then analyzed on a BD LSRII flow cytometer (BDBiosciences).

In Vivo Treatment of MM-Bearing Mice and Bioluminescence Imaging

MM.1 S and IM9 myeloma cells were retrovirally transduced withPinco-pGL3-luc/GFP virus expressing firefly luciferase, and GFP-positivecells were sorted using the aforementioned method, yielding MM.1S-GL3and IM9-GL3 cells, respectively. Male NSG mice were intravenouslyinjected with 8×10⁶ MM.1S-GL3 cells or 5×10⁵ IM9-GL3 cells in 400 μL ofPBS via tail vein on day 0 to establish a xenograft orthotopic MM model.On days 7 and 14 (MM.1S) or 21 (IM-9), the mice were intravenouslyadministered with 10×10⁶ effector cells, CS1-CAR-transduced T cells ormock-transduced control cells, in 400 mL of PBS via tail vein. Fiveweeks after inoculation with MM cells, the mice were intraperitoneallyinfused with D-luciferin (150 mg/kg body weight; Gold Biotechnology),anesthetized with isoflurane, and imaged using the In Vivo ImagingSystem (IVIS) with Living Image software (PerkinElmer).

Statistical Analysis

The unpaired Student t test was used to compare two independent groupsfor continuous endpoints if normally distributed. One-way ANOVA was usedwhen three or more independent groups were compared. For survival data,Kaplan-Meier curves were plotted and compared using a log-rank test. Alltests were two-sided. P values were adjusted for multiple comparisonsusing the Bonferroni method. A P value of less than 0.05 is consideredstatistically significant.

Results

Generation of Primary T Cells Expressing CS1-Specific CAR

A Pinco retroviral vector encoding a CS1-specific CAR (Pinco-CS1-CAR)was constructed, which consisted of anti-CS1 scFv, the hinge andtransmembrane regions of the CD8 molecule, the CD28 costimulatorysignaling moiety, and the cytoplasmic component of CD3 molecule (FIG.1A). Anti-CD3/CD28 antibody-activated primary T cells from a healthydonor were transduced with retroviral particles encoding CS1-CAR orempty vector (mock) and sorted for expression of GFP, which was encodedby the retroviral construct. To determine whether CS1-CAR wassuccessfully transferred, the sorted cells were lysed and subjected toimmunoblotting with an anti-CD3ζ mAb. As shown in FIG. 1B, in contrastwith the mock-transduced T cells, which only expressed endogenous CD3ζprotein, CS1-CAR-transduced T cells expressed the chimericCS1-scFv-CD28-CD3ζ fusion protein at the predicted size in addition tonative CD3ζ. Expression of CS1-CAR on the cell surface was demonstratedby staining transduced T cells with a goat anti-mouse Fab antibody thatrecognized the scFv portion of anti-CS1, which detected expression ofthe scFV on 70.3% of CS1-CAR-transduced T cells, whereas expressionremained almost undetectable on mock-transduced T cells (FIG. 1C).

Recognition of CS1⁺ Myeloma Cell Lines by CS1-Specific CAR T Cells

Surface expression of CS1 was evaluated in four commonly used myelomacell lines NCI-H929, IM9, MM.1S, and RPMI-8226 by flow cytometry, whichrevealed that CS1 protein was variably expressed in these cell lineswith much higher expression in NCI-H929, IM9, and MM.1S cells thanRPMI-8226 cells with minimal CS1 expression (FIG. 2A). As a negativecontrol, the transformed human kidney cell line, 293T, did not expressCS1 on its surface (FIG. 7A). To determine the capacity of CS1-CAR Tcells for recognition of myeloma cells that endogenously expressed CS1,IFN-γ, and IL-2 secretion was measured via ELISA in supernatants frommock-transduced T cells or CS1-CAR-transduced T cells in the presence orabsence of each myeloma cell line. Mock-transduced T cells andCS1-CAR-transduced T cells each alone produced negligible levels ofIFN-γ and IL-2 (FIGS. 2B and C); however, after exposure to NCI-H929 andIM9 cells expressing high levels of CS1, significantly greater amountsof IFN-γ and IL-2 proteins were secreted by CS1-CAR T cells but not bymock T cells. In response to MM.1S cells with high levels of CS1expression, CS1-CAR-transduced T cells also produced a higher amount ofIFN-γ than mock-transduced T cells (FIG. 2B) whereas, for unknownreasons, CS1-CAR-transduced T cells could not be triggered by this cellline to secrete higher levels of IL-2 than mock-transduced T cells (FIG.2C). In addition, compared with corresponding mock-transduced subsets ofT cells, both CD4⁺ (CD8) and CD8⁺ CS1-CAR T cells displayed increasedIFN-g secretion in response to NCI-H929 or MM.1S cells (FIG. 8A). ForRPMI-8226 cells with very low levels of CS1 expression, bothmock-transduced T cells and CS1-CAR-transduced T cells produced lowlevels of IFN-g and IL-2 that were comparable with background (FIGS. 2Band C). These findings suggest that, compared with mock-transduced Tcells, CS1-CAR-transduced T cells can more specifically recognize MMcells with high levels of endogenous CS1 expression, and become moreactivated after the recognition of these MM cells.

In Vitro Cytolytic Potency Against Myeloma Cells Triggered byCS1-Specific CAR

To determine whether enhanced recognition of CS1⁺ myeloma cells byCS1-CAR T cells could lead to more efficient tumor cell lysis, astandard 4-hour chromium-51 release assay was performed. NCI-H929, IM9,and MM.1S cells, which express high levels of CS1, were resistant tomock-transduced T-cell-mediated killing, even at E/T ratios as high as20:1; however, these cells were efficiently lysed by CS1-CAR T cells atall E:T ratios tested (FIG. 3A, left three). However, compared withmock-transduced T cells, the cytolytic activity of RPMI-8226 cellsexpressing low levels of CS1 could only be slightly augmented uponco-incubation with CS1-CAR-transduced T cells (FIG. 3A, right one).Degranulation and activation of T cells was further characterized byassessing expression of CD107a and CD69 in mock-transduced T cells andCS1-CAR-transduced T cells following incubation with or without NCI-H929myeloma cells which, as mentioned above, triggered a strong response inCS1-CAR T cells with respect to cytokine release and cytolytic activity.Consistent with the aforementioned data about cytokine release andcytolytic activity, degranulation and activation occurred to a greaterextent in CS1-CAR T cells than in mock T cells in response to NCI-H929cells, as evidenced by upregulation of surface coexpression of mobilizedCD107a and the activation marker, CD69 (FIG. 3B).

Moreover, compared with corresponding mock-transduced subsets of Tcells, both CD4⁺ (CD8⁻) and CD8⁺ CS1-CAR T cells exhibited increasedlevels of degranulation when stimulated by NCI-H929 or MM.1 S cells(FIG. 8B). In addition, using an intracellular staining approach,compared with mock-transduced T cells, CS1-CAR-transduced T cellsexpressed significantly higher levels of granzyme B, but not perforin,even in the absence of target cells (FIGS. 3C and D), suggesting thatgranzyme B may be predominantly involved in mediating the cytolyticactivity of CS1-redirected T cells. This finding is in line with aprevious report showing T cells grafted with a carcinoembryonicantigen-specific CAR incorporating a combined CD28-CD3ζ signaling moietyharbored elevated levels of granzyme B compared with unmodified T cells(Koehler H, et al. Cancer Res 2007 67:2265-73).

Forced Overexpression of CS1 in Target Cells Enhances Recognition andKilling by CS1-Specific CAR T Cells

The considerably stronger response in CS1-CAR T cells in terms ofcytokine release and cytotoxicity when stimulated by myeloma cellsexpressing high levels of CS1 prompted investigation of whether ectopicexpression of CS1 in myeloma cells with endogenously low levels of CS1expression could elicit an increase in cytokine release and cytolysis.To this end, RPMI-8226 myeloma cells with low levels of endogenous CS1expression were transduced with lentiviruses encoding human CS1 or PCDHempty vector as a mock-transduced control. The transduction efficiencywas monitored by detection of GFP protein encoded by the lentiviruses,and the percentage of GFP-positive cells was more than 90% by flowcytometric analysis. Overexpression of CS1 was confirmed by staining thesurface of the transduced cells with a PE-conjugated anti-CS1 antibody(FIG. 4A). Chromium-51 release assay indicated that forced CS1expression resulted in a discernible increase in the susceptibility ofRPMI-8226 cells to lysis by CS1-CAR-transduced T cells as opposed tomock-transduced T cells (FIG. 4B). Then, IFN-γ and IL-2 production wasassessed via ELISA, showing that, compared with mock-transduced T cells,CS1-CAR-transduced T cells produced significantly higher amounts ofIFN-γ and IL-2 in response to RPMI-8226 cells overexpressing CS1;meanwhile, there was only a moderate increase in IFN-γ secretion and nochange in IL-2 secretion when CS1-CAR T cells were co-cultured withempty vector-modified RPMI-8226 cells (FIGS. 4C and D). Likewise,overexpression of CS1 in CS1⁻ 293T, a transformed cell line, alsotriggered enhanced cytokine release and cytolysis by CS1-CAR T cells(FIG. 7B-7D). This was consistent with other reports on CAR T cellstargeting other tumor antigens (Sanchez C, et al. Prostate CancerProstatic Dis 2013 16:123-31; Chinnasamy D, et al. J Clin Invest 2010120:3953-68). These findings corroborated that increased recognition andkilling of target cells by CS1-CAR T cells occurred in a CS1-dependentmanner.

Improved Recognition and Killing of Primary Myeloma Cells by AutologousCS1-Specific CAR T Cells

To study the effects of CS1-specific CAR T cells in a more clinicallyrelevant context, it was investigated whether CS1-CAR-transducedautologous T cells could efficiently recognize and kill tumor cellsfreshly isolated from patients with myeloma. Like T cells from healthydonors, T cells from patients with relapsed myeloma were successfullyexpanded and manipulated to express CS1-CAR by retroviral infection, asmanifested by 60.7% of T cells staining positively with both anti-mouseFab and anti-human CD3 antibodies determined by flow cytometry (FIG.5A). Primary CD138⁺ myeloma cells from patients were isolated usingpositive magnetic selection, and primary myeloma cells were observed tobe uniformly positive for surface expression of CS1 using flow cytometry(FIG. 5B). By chromium-51 release assay, it was observed that myelomacells from patients were highly resistant to lysis by autologousmock-transduced T cells, but became susceptible to autologousCS1-CAR-transduced T cells even at a low (2.5:1) (E/T) ratio (FIG. 5C).In agreement with these cytotoxicity results, autologous CS1-CAR T cellsproduced significantly higher amounts of IFN-γ in response to myelomacells compared with autologous mock-transduced T cells (FIG. 5D). Thesefindings demonstrate that CS1-CAR-equipped T cells can efficientlyrecognize and eradicate myeloma cells in the autologous setting ex vivo.

CS1-Directed T Cells Suppress In Vivo Tumor Growth and Prolong Survivalof Tumor-Bearing Mice in Orthotopic Xenograft Myeloma Models

The therapeutic potential of CS1-CAR T cells was evaluated in anMM.1S-grafted NSG mouse model. Intravenous injection of MM.1S cells hasbeen widely used to establish a mouse xenograft model of MM, becausethis can lead to the engraftment in bone marrow and bone, as well asconsistent establishment of multifocal bone lesions, which closelyrecapitulates human MM (Mitsiades C S, et al. Cancer Cell 2004 5:221-30;Runnels J M, et al. J Biomed Opt 2011 16:011006). To facilitatemonitoring of tumor growth, MM.1S cells were engineered to express bothGFP and firefly luciferase by retroviral infection, and GFP⁺ cells weresorted and intravenously grafted into NSG mice to initiate tumor growth.These mice were then intravenously infused with mock-transduced T cells,CS1-CAR-transduced T cells or PBS. In agreement with the previousreports (Mitsiades C S, et al. Cancer Cell 2004 5:221-30; Runnels J M,et al. J Biomed Opt 2011 16:011006), bioluminescence imaging using IVISshowed that MM.1S-bearing NSG mice in the PBS-treated group developeddisseminated tumor lesions in skulls, vertebrae, pelvis, and femurs(FIG. 6A), and the majority of the mice displayed hind leg paralyses 5weeks after inoculation of tumor cells. Infusion of CS1-CAR T cellsremarkably reduced tumor burden as determined by bioluminescence imagingas well as prolonged the overall survival of MM.1 S-bearing NSG mice,whereas infusion of mock-infected T cells failed to result in efficienttumor eradication and improved survival of mice (FIGS. 6A and B).

To further validate the in vivo anti-MM capacity of CS1-CAR T cells, theimpact of CS1-CAR T cells was evaluated using an IM9-grafted NSG mousemodel. Similar data to those shown using MM.1S were observed.Bioluminescence imaging indicated that infusion of CS1-CAR-transduced Tcells could efficiently eradicate tumors established in IM9-bearingmice, whereas infusion of mock-transduced T cells failed to reduce tumorburden (FIG. 9A). Forty-four days after the initial treatment, a 100%survival rate was observed for IM9-bearing mice receiving CS1-CAR T-cellinfusion, whereas the survival rate was only 28.6% and 16.7% for controlmice receiving mock T cells and PBS, respectively (FIG. 9B).

Example 2: CS1-Specific Chimeric Antigen Receptor (CAR)-EngineeredNatural Killer Cells Enhance In Vitro and In Vivo Antitumor ActivityAgainst Human Multiple Myeloma

In this study, human NK cells were engineered to express a CAR that wasCS1 specific, and incorporated a CD28-CD3ζ co-stimulatory signalingdomain. The anti-MM function of these cells was evaluated in vitro andin an in vivo orthotopic xenograft mouse model of MM. The results showedthat the expression of the CS1-CAR could redirect NK cells tospecifically and efficiently eradicate CS1-expressing MM cells, both invitro and in vivo, and this eradication was CS1 dependent. The datasuggest that this CAR strategy is suitable for the development of aneffective NK cell-based immunotherapy as a means to treat patients withrefractory or relapsed MM. In addition, in contrast to CAR T cells, CARNK cells allow the use of allogeneic NK cell sources, which are lesslikely to cause and may even help to suppress graft-versus-host disease(Olson J A, et al. Blood 2010 115:4293-4301), while also potentiating anincrease in cytotoxicity due to mismatched killer immunoglobulin-likereceptors (KIRs) (Ruggeri L, et al. Science 2002 295:2097-2100).

Materials and Methods

Cell Culture

Human multiple myeloma cell lines L363 (German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany), IM9 [AmericanType Culture Collection (ATCC), Manassas, Va., USA] and U266 (ATCC) weremaintained in RPMI 1640 medium with 10% fetal bovine serum (FBS) (LifeTechnologies, Grand Island, N.Y., USA). Human IL-2-dependent NK celllines NK-92 (ATCC) and NKL were maintained in RPMI 1640 mediumsupplemented with 20% FBS and 150 IU/mL rhIL-2 (Hoffman-LaRoche Inc.,Nutley, N.J., USA). 293T (ATCC), and the phoenix packaging cell line wasmaintained in DMEM medium with 10% FBS. Primary CD138⁺ MM cells wereisolated from bone marrow aspirate of MM patients using the EASYSEPHuman Whole Blood and Bone Marrow CD138 Positive Selection Kit (StemCellTechnologies, Vancouver, BC, Canada) according to the manufacturer'sprotocol. All human work was approved by The Ohio State UniversityInstitutional Review Board.

Mice

Six- to eight-week-old NOD.Cg-prkdcscid IL2rgtm1Wjl/szJ (NSG) mice wereobtained from Jackson Laboratories (Bar Harbor, Me., USA). All animalwork was approved by The Ohio State University Animal Care and UseCommittee. Mice were monitored frequently for MM disease progression,and killed when they were moribund with the symptoms of hindlimbparalysis, lethargy, and obvious weight loss.

Generation of Anti-CS1 CAR Lentiviral Construct

The CS1-scFv fragment, amplified from the hybridoma cell line Luc90, wasfused with a sequence encoding a Myc tag immediately following theCS1-VL-encoding sequence. The fused DNA sequences were incorporated withCD28-CD3ζ that was incised from a retroviral vector. The entireCS1-scFv-myc tag-CD28-CD3ζ fragment was ligated into a lentiviral vectordesignated as PCDH-CMV-MCS-EF1-copGFP (PCDH, System Biosciences,Mountain View, Calif., USA) to generate a PCDH-CS1-scFv-myctag-CD28-CD3ζ (PCDH-CS1-CAR) construct.

Lentivirus Production and Transduction of NK Cells

To produce VSVG-pseudotyped lentiviral supernatant, 293T cells culturedin DMEM media (Invitrogen) were co-transfected withPCDH-CS1-scFv-CD28-CD3c or the PCDH control vector (to generate virusfor mock infection with the empty vector) together with the packagingconstructs, pCMV-VSVG and pCMV-dr9, using calcium phosphate transfectionreagent (Promega, Madison, Wis., USA). After 24 h, the DMEM media wasreplaced with RPMI-1640 media containing 20% FBS. 48 h aftertransfection, conditioned medium containing lentivirus was harvested andfiltered through a 0.45 m filter unit (Milliopore, Billerica, Mass.,USA) to remove cell debris. Viral infection was performed in 6-wellplates using 2×10⁶ NK-92 or NKL cells in a total volume of 2 mL oflentiviral supernatant containing 8 μg/mL polybrene (Sigma-Aldrich, St.Louis, Mo., USA) and 450 IU/mL rhIL-2. Cells were centrifuged at 1,800rpm at 32° C. for 45 min, then plates were placed in an incubator at 37°C. for 2 h. Infection was then repeated a second time on the same day,and one additional time the following day. After the third transduction,cells were maintained in RPMI 1640 media supplemented with 20% FBS and150 IU/mL rhIL-2 at 37° C. Transduced NK cells were enriched by tworounds of cell sorting using a FACS Aria II cell sorter (BD Biosciences,San Jose, Calif., USA). Positive cells were selected based on expressionof green fluorescence protein (GFP) surface marker, which was encoded inthe PCDH vector

Generation of a U266 Cell Line Stably Expressing CS1

Human CS1 coding sequences were amplified from cDNA isolated from IM9cells via PCR, then subcloned into a PCDH lentiviral vector to generatea PCDH-CS1 construct. Lentivirus production and infection of U266 cellswere performed using the methods described above. GFP-positive cellswere then sorted using an FACS Aria II cell sorter (BD Biosciences, SanJose, Calif., USA).

Immunoblotting Analysis

Cells were washed with PBS and directly lysed in laemmli buffer. Lysateswere electrophoretically separated on a 4% to 15% gradient SDS-PAGE gel(Bio-Rad Laboratories, Hercules, Calif., USA) and transferred to anitrocellulose membrane (EMD Millipore, Billerica, Mass., USA). Themembrane was blocked with 5% milk in Tris Buffered Saline (TBS)supplemented with 0.1% Tween 20. Mouse anti-human CD3ζ chain monoclonalantibody (BD Pharmingen, San Diego, Calif., USA) was diluted 1:1,000with 5% milk in TBS supplemented with 0.1% Tween 20, and this antibodysolution was added to react with the membrane overnight. The membranewas then washed three times in TBS supplemented with Tween 20. TheHRP-conjugated secondary antibody (GE Healthcare Biosciences,Pittsburgh, Pa., USA) was diluted 1:5,000 with 5% milk in TBSsupplemented with 0.1% Tween 20 and added to the membrane to stand for 1h. The membrane was again washed four times in TBS supplemented withTween 20, and an enhanced chemiluminescence reagent (ECL; GE HealthcareBiosciences) was added for 1 min. The blot was then exposed to film forvarious lengths of time to generate a properly exposed image.

Flow Cytometry

To analyze surface expression of CS1-CAR, a single cell suspension oftransduced NK cells was incubated for 1 h at 4° C. with an anti-Myc tagmouse mAb 9E10 (Sigma-Aldrich). Cells were washed twice with PBS andthen incubated for 30 min at 4° C. with PE-conjugated rat anti-mouseIgG1 secondary antibody (BD Pharmingen). Surface expression of CS1 andCD138 on myeloma cells was examined by FACS analysis using a BD LSRIIanalyzer after cells were stained with PE-conjugated mouse anti-CS1 mAb(eBiosciences, San Diego, Calif., USA) and APC-conjugated mouseanti-CD138 mAb (BD Pharmingen). Data analysis was carried out usingFLOWJO software (Tree Star Inc., Ashland, Oreg., USA).

Cytotoxicity Assay

For detection of NK cell-mediated lysis, MM target cells were labeledfor 1.5 h with 100 mCi chromium-51 (⁵¹Cr), washed four times withregular RPMI media, and adjusted to a concentration of 5,000 cells perwell in 100 μl volume of a 96-well V-bottom microtiter plate.FACS-enriched mock- or CS1-CAR-transduced NK cells were added in 100 μlvolume into triplicate wells at various effector to target (E:T) ratios.After 4 h of incubation at 37° C., 100 μl of supernatant was harvestedfrom each well, and transferred to scintillation vials containing aliquid scintillation cocktail (Fisher scientific, Pittsburgh, Pa., USA)so that release of ⁵¹Cr could be measured on a TOPCOUNT counter(Canberra Packard, Meriden, Conn., USA). To determine maximal ⁵¹Crrelease, target cell suspension was incubated with 100 μl of SDS.Percentage of specific lysis was calculated using the standard formula:100×(cpm experimental release−cpm spontaneous release)/(cpm maximumrelease−cpm spontaneous release).

Interferon-γ Release Assay

Myeloma target cells were co-cultured with NK effector cells in 96-wellV bottom plates for 24 h. In all, 2.5×10⁵ myeloma cell line cells or1.0×10⁵ primary myeloma cells were incubated with 2.5×10⁵ or 5.0×10⁵ NKcells, respectively. Cell-free supernatants were assayed forinterferon-γ (IFN-γ) secretion by enzyme-linked immunosorbent assay(ELISA) using a kit from R&D Systems (Minneapolis, Minn., USA) accordingto the manufacturer's protocol. Data depicted in Figures represent meanvalues of triplicate wells from one of three representative experimentswith similar results.

An Orthotopic MM Mouse Model and In Vivo Treatment of MM-Bearing Miceand Bioluminescence Imaging

IM9 cells were retrovirally transduced with Pinco-pGL3-luc/GFP virusexpressing firefly luciferase as previously described (He S, et al.Blood 2013 121:4663-4671). GFP-positive cells were sorted using an FACSAria II cell sorter (BD Biosciences), and were designated as ‘IM9-Luc’cells. Then, six- to eight-week-old male NSG mice were intravenously(i.v.) injected with 0.5×10⁶ IM9-Luc MM cells in 400 μl ofphosphate-buffered saline via tail vein on day 0 to establish axenograft orthotopic MM model. Beginning on day 7, the mice were i.v.injected with 5×10⁶ effector cells, that is, CS1-CAR NK-92 cells ormock-transduced control cells, in 400 μl of phosphate-buffered salineonce every 5 days (five times in total). Four weeks after IM9-Lucinoculation, the mice were intraperitoneally (i.p.) infused withD-luciferin (150 mg/kg body weight; Gold Biotechnology, St. Louis, Mo.,USA), anesthetized with isoflurane, and imaged using an In Vivo ImagingSystem (IVIS-100, Perkin-Elmer, Waltham, Mass., USA) with the LivingImage software (Perkin-Elmer).

Immunohistochemical Analysis

Spinal vertebrae were fixed in 10% buffered formalin phosphate anddecalcified in saturated EDTA, and then embedded in paraffin.Five-micron thick sections were stained with hematoxylin and eosin (H&E)for histological examination. The sections were immunostained foridentification of human MM cells with mouse anti-human CD138 mAb (1:50dilution; Thermo-Scientific, Waltham, Mass., USA) following standardimmunohistochemistry staining procedures. Horseradishperoxidase-conjugated anti-mouse IgG was used as a secondary antibody,followed by a peroxidase enzymatic reaction.

Statistics

Unpaired Student's t test was utilized to compare two independent groupsfor continuous end points if normally distributed. One-way ANOVA wasused when three or more independent groups were compared. Fornon-normally distributed end points, such as in vivo bioluminescenceintensity, a Kruskal-Wallis test was utilized to compare the median ofNK-92-CS1-CAR to NK-92-EV and phosphate-buffered saline groups. Forsurvival data, Kaplan-Meier curves were plotted and compared using alog-rank test. All tests are two-sided. P-values were adjusted formultiple comparisons by the Bonferroni method. A P-value of <0.05 isconsidered as statistically significant.

Results

Generation of NK-92 and NKL NK Cells Expressing CS1-CAR

A specific CS1-CAR construct was generated with a PCDH lentiviral vectorbackbone, sequentially containing a signal peptide (SP), a heavy chainvariable region (VH), a linker, a light chain variable region (VL), aMyc tag, a hinge, CD28 and CD3ζ (FIG. 13A). NK-92 and NKL NK cell lineswere transduced with the CAR construct and then sorted for expression ofGFP, a marker expressed by the vector. Western blotting of the sortedcells demonstrated that CS1-CAR was successfully introduced andexpressed, as evidenced by the expression of the chimeric CS1-scFvreceptor containing CD3ζ in both NK-92 and NKL cell lines transducedwith the CAR construct rather than with the control vector (FIG. 13B).Moreover, a flow cytometric analysis after anti-Myc Ab surface stainingindicated that CS1-CAR was expressed on the surface of both NK-92 andNKL cells transduced with the CS1-CAR construct (FIG. 13C).

CS1-CAR-Modified NK Cells More Effectively Eradicate CS1⁺ MM Cells, butnot CS1⁻ Cells, In Vitro in Comparison with Mock-Transduced NK Cells

After generating the CS1-CAR NK cells, it was determined whether theyselectively kill CS1 better than CS1 MM cells. For this purpose, it wasfirst confirmed that IM9 and L363 MM cell lines constitutively expressedCS1 protein on their surface, while expression of CS1 was negligible inU266 MM cells (FIG. 14A). Next, a 4-h chromium-51 release assayindicated that, compared with mock-transduced NK-92 cells, NK-92 cellstransduced with CS1-CAR were significantly enhanced in their ability tokill CS1⁺ IM9 and L363 cells (FIGS. 14B and 14C, left panels). Similardata were observed in experiments repeated using NKL cells transducedwith CS1-CAR (FIGS. 14B and 14C, right panels). However, both theCS1-CAR- and mock-transduced NK-92 or NKL cells were similar in theirlow levels of cytotoxicity against CS1U266 myeloma cells (FIG. 14D). Inaddition, forced expression of CS1-CAR did not induce obvious apoptosisin NK-92 or NKL cells as determined by analyses of 7AAD/AnnexinV-staining using flow cytometry (FIG. 20), suggesting that CS1-CARexpression did not cause cytotoxicity to the NK-92 or NKL cellsthemselves. Similarly, CS1-CAR expression in purified primary human NKcells augmented their cytotoxicity against CS1⁺ IM9 myeloma cells.

CS1-CAR-Modified NK Cells Secrete More IFN-γ than Mock-Transduced NKCells do after Exposure to CS1⁺ MM Cells

The signaling domain of the CD28 co-stimulatory molecule, which wasincluded in the CAR construct, may enhance activation after recognitionof the CS1 scFv with the CS1 antigen on the surface of MM cells.Therefore, the inclusion of this signaling domain may have the capacityto activate NK cells not only to have higher cytotoxicity, but also toproduce more IFN-γ, the latter of which is also important for tumorsurveillance and activation of CD8⁺ T cells and macrophages(Martin-Fontecha A, et al. Nat Immunol 2004 5:1260-1265; Tu S P, et al.Cancer Res 2011 71:4247-4259; Ma J, et al. Cell Mol Life Sci 200360:2334-2346). To test this, CS1-CAR-modified or control-engineeredeffector NK cells were either cultured alone or co-cultured with CS1⁺myeloma cells including the IM9 and L363 MM cell lines. After 24 h, theIFN-γ production was measured by ELISA. As shown in FIG. 15, bothCS1-CAR-modified and mock-transduced NK-92 or NKL cells spontaneouslyproduced low or negligible levels of IFN-γ when incubated alone.Co-culture with CS1 MM tumor cells (IM9 or L363) induced IFN-γ in bothCS1-CAR and mock-transduced NK-92 or NKL cell lines; however,significantly higher levels of IFN-γ were produced by CAR-modified NK-92or NKL cells than by mock-transduced NK-92 (FIGS. 15A and 15B, leftpanels) or NKL cells (FIGS. 15A and 15B, right panels). When co-culturedwith the CS1 MM cell line, U266, both mock-transduced andCS1-CAR-transduced NK-92 cells but not the transduced NKL cells producedhigher levels of IFN-γ than corresponding cells that had not beenco-cultured with U266 cells (FIG. 15C). This suggests that a uniqueinteraction between NK cell receptors on NK-92 cells and their ligandson U266 cells may induce CS1-independent IFN-γ production by NK-92cells. Moreover, CS1-CAR-transduced NK-92 and NKL cells failed toproduce more IFN-γ than corresponding mock-transduced NK-92 and NKLcells when they were co-cultured with U266 cells (FIG. 15C). Theseresults are in agreement with the aforementioned cytotoxicity data, andtogether indicate that modification with CS1-CAR can dramaticallyenhance NK cell effector functions, in terms of both cytotoxicity andIFN-γ production, in response to CS1⁺ but not to CS1⁻ myeloma cells.

Enforced CS1 Expression in U266 Cells Enhances Cytotoxicity and IFN-γProduction of NK-92-CS1-CAR Cells

It was next explored whether this enhanced activity of CS1-CAR NK cellsrelies on CS1 antigen expression on MM cells. The aforementionedobservation—that the introduction of CS1-CAR conferred NK-92 cells withincreased cytotoxic activity and enhanced IFN-g production in responseto CS1⁺ myeloma cells, but not to CS1⁻ U266 myeloma cells-promptedinvestigation of whether CS1 overexpression in U266 cells is sufficientto change the sensitivity of U266 cells to NK-92-CS1-CAR cells. For thispurpose, CS1 was ectopically expressed in U266 cells by lentiviralinfection. Flow cytometric analysis confirmed that CS1 protein wassuccessfully expressed on the surface of the U266-CS1 cells (FIG. 16A).Chromium-51 release assay indicated that, when compared withmock-transduced NK-92 cells, there was a significant increase in thecytotoxic activity of CS1-CAR-transduced NK-92 cells toward U266 cellsoverexpressing CS1 (FIG. 16B). Likewise, compared with parallelco-cultures containing mocktransduced NK-92 cells, NK-92-CS1-CAR cellsco-cultured with U266 cells overexpressing CS1 secreted significantlyhigher levels of IFN-g (FIG. 16C). However, consistent with data inFIGS. 14D and 15C, there was no difference in cytotoxicity and IFN-γsecretion between NK-92-CS1-CAR cells and mock-transduced NK-92 cellswhen they were incubated with U266 cells transduced with an empty vectorcontrol (FIGS. 16B and 16C). These results suggested that the increasedrecognition and killing of myeloma cells by NK-92-CS1-CAR cells occursin a CS1-dependent manner.

Phenotypic Characterization of NK-92-CS1-CAR Cells

It was next investigated whether the expression of a CS1-specific CARcould change the NK cell phenotype. Flow cytometry was used to compareexpression of antigens on the surface of CS1-CAR-transduced andmock-transduced NK-92 cells, following culture in the presence orabsence of IM9 myeloma cells. As shown in FIG. 17A, there was nodifference between CS1-CAR- and mock-transduced NK-92 cells, whethercultured in the presence or absence of IM9 cells, in the expression ofNK cell receptors including NKp30, NKp46, NKG2C and NKG2D. Expression ofthe NK cell activation markers, CD6928 and HLA-DR (Phillips J H, et al.J Exp Med 1984 159:993-1008; Spits H, et al. Immunity 2007 26:11-16) wasalso assessed. Recognition of IM9 cells did not elicit CD69 expressionon mock-transduced NK-92 cells, yet induced a moderate, but significant,increase in CD69 expression on the surface of CS1-CAR-transduced NK-92cells (FIG. 17A). Interestingly, co-incubation with IM9 cells caused adramatic increase in the expression of HLA-DR in both CS1-CAR-transducedand mock-transduced NK-92 cells. In the absence of IM9 target cells,there was no obvious difference in HLA-DR expression betweenCS1-CAR-transduced and mock-transduced NK-92 cells; however, uponstimulation with IM9 cells, the expression of HLA-DR becamesignificantly higher in NK-92-CS1-CAR cells than in mock-transducedNK-92 cells. Thus, the increase in the activation markers, especiallyHLA-DR, expressed on NK-92-CS1-CAR cells may have occurred in connectionwith the enhanced cytotoxicity and IFN-γ production by these cells whenthey are exposed to CS1 MM cells. Using intracellular staining, whencompared with mock-transduced NK cells, NK-92-CS1-CAR cells hadsignificantly higher levels of perforin and granzyme B expression, evenin the absence of MM tumor cells (FIGS. 17B and 17C). This is consistentwith a previous report regarding the elevated expression of granzyme Bin CAR T cells (Koehler H, et al. Cancer Res 2007 67:2265-2273), andalso consistent with the fact that perforin and granzyme B expressionare generally correlated with cytotoxic activity of NK cells (KrzewskiK, et al. Front Immunol 2012 3:335).

CS1-CAR-Transduced NK-92 Cells More Effectively Recognize and KillNK-Resistant Primary MM Cells Ex Vivo

To make the findings more clinically relevant, it was investigatedwhether CS1-CAR-modified NK-92 cells also harbored enhanced cytolyticactivity and IFN-γ production when recognizing primary MM cells ex vivo.Flow cytometry was used to assess surface expression of CS1 on primaryCD138⁺ magnetic bead-selected MM cells from MM patients (FIG. 18A). Inaccordance with the previous report, showing that CS1 protein was highlyexpressed on CD138 magnetic bead-purified MM patient cells (Hsi E D, etal. Clin Cancer Res 2008 14:2775-2784; Tai Y T, et al. Blood 2008112:1329-1337), CS1 protein was indeed uniformly expressed on thesurface of primary MM cells (FIG. 18A). By chromium-51 release assay,primary myeloma cells freshly isolated from MM patients were shown to behighly resistant to NK-92 cell-mediated lysis even at E:T ratios as highas 40:1 and 20:1; however, this resistance could be partially overcomeby NK-92 cell expression of CS1-CAR, which resulted in a dramaticincrease in eradication of primary myeloma cells (FIG. 18B). In linewith the cytotoxicity result, after 24 h co-culture with primary myelomacells, CS1-CAR-transduced NK-92 cells also secreted significantly higherlevels of IFN-γ than mock-transduced NK-92 cells (FIG. 18C).

CS1-CAR-Transduced NK-92 Cells Inhibit MM Tumor Growth and ProlongSurvival of Tumor-Bearing Mice in an Orthotopic Xenograft MM Model

To further address the potential therapeutic application ofNK-92-CS1-CAR cells, their antitumor activity was examined inIM9-xenografted NSG mice. An IM9 cell line expressing firefly luciferase(IM9-Luc) was generated by retrovirally transducing IM9 cells with virusexpressing firefly luciferase, then performing GFP-based cell sorting.The expression of full-length firefly luciferase mRNA was confirmed byRT-PCR. Like typical myeloma cells, IM9-Luc cells expressed CD138protein on their surface. In agreement with a previous report (FranciscoJ A, et al. Cancer Res 2000 60:3225-3231), IM9-Luc-bearing NSG micedisplayed disseminated disease, manifested by hindlimb paralysis andmotor ataxia. Histological examination of spinal vertebrae in a mousedisplaying hindlimb paralysis showed the presence of numerous tumorcells and osteolytic lesions in bone tissue (FIG. 19A, left).Immunohistochemical staining with human-specific anti-CD138 antibodyfurther confirmed the presence of tumor cells (FIG. 19A, right).Bioluminescence imaging was used to monitor the IM9-Luc tumor growth. Asshown in FIGS. 19B and 19C, and in agreement with the in vitrocytotoxicity data, comparing the mice who later received injections withmock-transduced control cells, IM9-Luc tumors were significantlysuppressed in mice who instead later were administered NK-92-CS1-CARcells. Moreover, treatment with NK-92-CS1-CAR cells significantlyprolonged the survival of mice bearing IM9-Luc tumors as compared withtreatment with the mock-transduced NK-92 control cells (FIG. 19D). Ofnote, when NK-92-CS1-CAR cells or mock-transduced NK-92 cells weresimilarly administered, but without i.v. injection of IM9-Luc cells,mice did not develop disseminated disease or die.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method of providing an anti-tumor immunity in asubject with multiple myeloma (MM), the method comprising administeringto the subject an effective amount of an immune effector cellgenetically modified to express a chimeric antigen receptor (CAR)wherein the CAR comprises a CS1 antigen binding domain, a costimulatorysignaling region, and an intracellular signaling domain, therebyproviding an anti-tumor immunity in the subject; wherein the CS1 antigenbinding domain comprises a single-chain variable fragment (scFv) of anantibody that specifically binds CS1.
 2. The method of claim 1, whereinthe immune effector cell is selected from the group consisting of a Tcell, a Natural Killer (NK) cell, and a cytotoxic T lymphocyte (CTL). 3.The method of claim 1, wherein the costimulatory signaling regioncomprises the cytoplasmic domain of a costimulatory molecule selectedfrom the group consisting of CD28 and 4-1BB.
 4. The method of claim 1,wherein the CAR polypeptide is defined by the formula:SP-CS1-HG-TM-CSR-ISD; wherein “SP” represents a signal peptide, wherein“CS1” represents a CS1 antigen binding domain, wherein “HG” representsan optional hinge domain, wherein “TM” represents a transmembranedomain, wherein “CSR” represents a co-stimulatory signaling region,wherein “ISD” represents an intracellular signaling domain, and wherein“-” represents a bivalent linker; and wherein the CS1 antigen bindingdomain comprises a single-chain variable fragment (scFv) of an antibodythat specifically binds CS1.
 5. The method of claim 1, wherein theintracellular signaling domain comprises a CD3 zeta (CD3) signalingdomain.
 6. The method of claim 3, wherein the costimulatory molecule isCD28.
 7. The method of claim 3, wherein the costimulatory molecule is4-1BB.
 8. The method of claim 1, wherein the subject is a human.
 9. Themethod of claim 1, wherein the CS1 antigen binding domain consists of asingle-chain variable fragment (scFv) of an antibody that specificallybinds CS1.